Rod and cone photoreceptors in the vertebrate retina provide the organism with two complementary signaling cells that support the full behavioral range of the visual system. In general, the photoresponse of rods is slower in time course, more sensitive to light and adapts over a more restricted range of intensities than that of cones. The long-term objective of this research is to understand the molecular mechanisms of phototransduction in the retinal photoreceptors, and to explain the functional differences between the two receptor types. Phototransduction arises from the activation by light of a linear sequence of enzymatic reactions, whose end point is the lowering of cytoplasmic cGMP concentration and closure of cGMP gated ion channels. In parallel with the changes in cGMP, there also occurs a lowering of cytoplasmic Ca2+ concentration. The change in Ca2+ is not essential; phototransduction occurs even in its absence. However, the Ca2+ changes impose regulation on the enzymatic cascade that is critical in the normal function of the cells. In particular, some of the functional differences between rods and cones arise from differences in the homeostasis of cytoplasmic Ca2+ in the outer segment and the modulatory effect of these changes. The specific aims of this proposal are to investigate in detail the mechanisms that regulate cytoplasmic Ca2+ in the outer segments and to determine the magnitude and time course of the light-dependent changes in Ca2+. The investigators will achieve these aims through biophysical studies in which the membrane current and cytoplasmic Ca2+ concentration will be measured simultaneously in isolated cone photoreceptors. They will investigate the functional properties of the pathways through which Ca2+ enters and leaves the cone outer segment. They will study the characteristics of the Ca2+ buffering sites in the outer segment. Among the functions that Ca2+ modulates is the sensitivity to cGMP of the cGMP-gated ion channels. This modulation is very different in rods and cones, and they propose to investigate it in detail in intact cone outer segments. To do so they will take advantage of a new preparation they have developed in which membrane currents can be measured in intact cone outer segments while controlling its cytoplasmic composition. The Ca2+-dependent modulation arises from the activity of an unidentified Ca2+-binding protein. They propose to identify, characterize and clone this molecule. While the differences in Ca2+ homeostasis are important, they are not sufficient to explain the functional differences between rods and cones. Theoretical and experimental work suggest that the rates at which the enzymes of the transduction cascade return to their dark state are faster in cones than in rods. They propose to test this hypothesis directly through studies of the kinetics of the membrane currents in single cone outer segments.